Project Abstract Streptococcus mitis and Streptococcus oralis normally colonize the human mouth but can also cause infections. S. mitis and S. oralis are common causes of invasive disease, including bacteremia, particularly in immunocompromised patients. As a result, they are primary pathogens for development of infective endocarditis (IE). Antibiotic resistance in S. mitis and S. oralis is rising, resulting in fewer treatment options and necessitating a deeper understanding of the basic physiology of these organisms. Despite their significance, the genes that empower S. mitis and S. oralis to proliferate in the bloodstream and on heart valves have not been elucidated. Further, infection isolates are poorly represented in genome sequencing analyses, therefore it is unknown whether these strains possess unique features that enhance their ability to proliferate in the bloodstream and on heart valves. The ramification is that some patients may be at higher risk for infection with S. mitis or S. oralis because of the strains in their microbiomes; identification of these strain characteristics along with patient risk factors for having these strains would enable more effective prophylaxis and prompt treatment. The research proposed here uses genome sequencing and mutagenesis approaches to investigate genetic mechanisms for virulence in these bacteria. In Aim 1, bacteremia and endocarditis isolates obtained from retrospective collection will be sequenced and compared with oral isolates using phylogenomics approaches. Aim 1 addresses two goals: A. Test the hypothesis that specific genetic features are associated with S. mitis and S. oralis infection. B. Define genomic differences between S. mitis and S. oralis. Patient characteristics will also be correlated to determine whether infection outcome is more associated with organism virulence versus host characteristics. Aim 2 will use targeted mutagenesis and transposon mutagenesis with sequencing (Tn-Seq) to identify genes required for S. mitis and S. oralis growth in blood and simulated endocardial vegetations. At completion, these Aims will [1] clarify what it means to be S. mitis or S. oralis, potentially identifying genes or phenotypes rapidly discriminatory for the two species; [2] determine whether high-risk lineages exist that are enriched with pathogenic and/or antibiotic resistance traits; and [3] begin to address whether infectivity is a core characteristic of S. mitis and S. oralis, enhanced by sequence variation and horizontal gene transfer, and/or is virulence more dependent upon individual host characteristics.